The completion of human genome project has catalyzed advances in proteomics to investigate cellular function at the protein level. In particular, increasingly sophisticated techniques have been rapidly developed for discovering disease biomarkers via large-scale differential profiling. The recognition that every disease induces a specific pattern of change in proteomic microenvironments indicates important clinical implications on the early detection and progression of disease. Although plasma, urine, and saliva are readily available samples whose protein content reflects the environment encountered by the blood during its journey through tissues and the circulatory system, the body fluid-derived proteomes are complex, with a wide and dynamic range in protein abundance that imposes extreme analytical difficulties for medical studies or clinical diagnoses. With the advent of a growing number of candidate protein biomarkers for disease diagnosis, the development of sensitive techniques with great potential to monitor disease onset is urgently needed for the next phase of targeted proteomics.
The detection and diagnosis of disease in the clinical setting primarily depends on immunoassays based on antibody-antigen interactions. The most widely used of all the methods, enzyme-linked immunosorbent assay (ELISA), offers both specificity and sensitivity. Alternatively, protein chip-based approaches are increasingly used in clinical diagnosis, because the array format can be easily adapted to miniaturization, multiplexing and high-throughput. However, these traditional immunological methods are inconvenient and time-consuming because enzymes or fluorescent reagents have to be labeled. Fluorescence measurements also may have high background, leading to false positives, and produce photobleaching, leading to false negatives (see, e.g., Graham et al., Trends Biotechnol., 2004, 22:455-462, herein incorporated by reference).
Recent developments in mass spectrometry have greatly expanded the possibility of characterizing unknown proteins in proteomic research. Mass spectrometry is especially suitable for the direct detection of proteins, which enhances specificity without the use of fluorescent or radioactive labels. This approach offers greater flexibility in the selection of bioactive probes. Among these developments, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become one of the primary techniques for protein identification due to its high sensitivity, tolerance to impurities, and high speed. Despite these advantages, the simultaneous characterization of hundreds to thousands of proteins in complex media still remains a challenge due to the suppression effect (see Wulflkuhle et al., Nat. Rev. Cancer, 2003, 3:267-275, herein incorporated by reference). Recently, surface-enhanced laser desorption/ionization (SELDI), has evolved rapidly as a new frontier for biomarker discovery and clinical diagnoses based on proteomic pattern analysis (see, Petricoin et al., Proteome Res. 2004, 3:209-217, herein incorporated by reference). Despite its advantages of high sensitivity and high throughput, the pattern recognition platform unfortunately suffers from laboratory-to-laboratory variance due to differences in sample handling and analysis software (see, Diamandis et al., Mol. Cell. Proteomics, 2004, 3:367-378, herein incorporated by reference).
As an alternative to the above approaches, MALDI MS can be combined with a biologically active probe to rapidly and specifically target proteins of interest. This targeted approach can accelerate research for class-specific proteins or biomarkers (Bundy et al., 2001, 73:751-757; Min et al., Nat. Biotechnol., 2004, 22:717-723; Warren et al., Anal. Chem., 2004, 76:4082-4092; and Zhang et al., Angew. Chem. Int. Ed., 2005, 44:615-617; all of which are herein incorporated by reference). Several analytical affinity capture techniques have been developed in the field of biological mass spectrometry. The research group of Hutchens et al. was one of the first to demonstrate MS-based affinity capture by immobilization of “bait” DNA on agarose beads for direct MALDI MS analysis of targeted proteins from complex biofluids (Hutchen et al., Mass Spectrom 1993, 7:576-580, herein incorporated by reference). The concept was further tailored by Nelson and coworkers to develop a mass spectrometric immunoassay (MSIA) (Nelson et al., Anal. Chem. 1995, 67:1153-1158, herein incorporated by reference). They used affinity pipette tips to selectively retrieve proteins from biological solutions, demonstrating high-throughput quantitative protein analysis as well as screening of heterogeneous glycan structures in plasma proteins (Nedelkow et al., Anal. Chem., 2004, 76:1733-1737; and Kiernan et al., Proteomics, 2004, 4:1825-1829, both of which are herein incorporated by reference). Variations of the biologically active probes for affinity mass spectrometry include the assay of direct desorption/ionization on silicon (DIOS) (Wei et al., Nature, 1999, 399:243-246, and Zou et al., Angew. Chem. Int. Ed. 2002, 41:646-648, both of which are herein incorporated by reference) and self-assembled monolayers (SAMs) (Brockman et al., Anal. Chem. 1995, 67, 4581-4585; and Su et al., Angew. Chem. Int. Ed. 2002, 41:4715-4718, both of which are herein incorporated by reference). Despite the rapid evolution of efficient chip-based or microbead-based assays for biomedical research, protein chip technologies face two main technical challenges. First, the physical and chemical properties of the chip surface may denature/alter the native three-dimensional structure of proteins, raising the possibility of disrupted bait-target protein interactions. Secondly, the requirement of specialized immobilization chemistry for surface engineering and/or specialized instruments limit the general application of these protein assay technologies in the general scientific community. Therefore, what is needed is a detection assay and associated compositions that avoid these problems.